nature publishing group Clinical Investigation Articles

Age-specific onset and distribution of the natural anticoagulant deficiency in pediatric thromboembolism

Masako Ichiyama1, Shouichi Ohga2, Masayuki Ochiai1, Koichi Tanaka1, Yuka Matsunaga1, Takeshi Kusuda1, Hirosuke Inoue1, Masataka Ishimura3, Tomohito Takimoto3, Yui Koga4, Taeko Hotta4, Dongchon Kang4 and Toshiro Hara1,3

Background: The early diagnosis of inherited throm- protein S (PS), and antithrombin (AT) deficiency, as well as bophilia in children is challenging because of the rarity and G1691A (FVL) and (FII) hemostatic maturation. variants (4). The high incidence of VTE in Caucasians (5) is Methods: We explored (PC), protein S (PS), explained by the fact that FVL and FII G20210A carriers were and antithrombin (AT) deficiencies in 306 thromboembolic found in 20–60% of adult VTE patients in Caucasian but not patients aged ≤20 y using the screening of plasma activity and Asian ancestries (6–9). PC, PS, and AT deficiencies share the genetic analysis. lower prevalence than FVL and/or FII G20210A carriers, but Results: Reduced activities were determined in 122 patients the higher risk of the first and recurrent VTE than the other (40%). Low PC patients were most frequently found in the low- . On the other hand, the diagnosis is challeng- est age group (0–2 y, 45%), while low PS or low AT patients ing during infancy and early childhood, because the references were found in the highest age group (16–20 y; PS: 30% and of increasing anticoagulant activity range widely until ado- AT: 20%). Genetic study was completed in 62 patients hav- lescence (10,11). The true effect of the natural anticoagulant ing no other causes of thromboembolism. Mutations were deficiencies on the development of pediatric thromboembo- determined in 18 patients (8 PC, 8 PS, and 2 AT genes). Six of lism remains elusive because of the rare occurrence and the eight patients with PC gene mutation were found in age 0–2 y elaborative genetic screenings. (75%), while six of eight patients with PS gene mutation were To clarify the clinical impact of the inherited deficiency of in 7–20 y. Two AT gene–mutated patients were older than 4 y. natural anticoagulant on children, we conducted the genetic Four PC-deficient and two PS-deficient patients carried com- analysis of PC, PS, and AT deficiency after the screening of pound heterozygous mutations. All but one PC gene–mutated each activity for pediatric patients with thromboembolism patient suffered from intracranial thromboembolism, while over 20 y in a single institution. The results demonstrated the PS/AT gene–mutated patients mostly developed extracranial distinct presentation of PC and PS deficiencies in infants and venous thromboembolism. children, respectively, reflecting the genotype of adult Japanese Conclusion: Stroke in low PC infants and deep vein throm- patients. bosis in low PS/AT school age children could be targeted for genetic screening of pediatric thrombophilias. RESULTS Distribution of Thromboembolic Patients Of 306 patients aged ≤20 y (male: female 1:1.14), 186 suffered hromboembolism is a multifactorial disease involving from intracranial lesions including ischemic and/or hemor- Tgenetic predispositions, underlying disorders, and var- rhagic strokes. The extracranial lesions consisted of renal vein ied triggers including infection or injury. Vascular, circula- (n = 7), fulminans (n = 3), deep vein tory, and hemostatic conditions are distinctively associated thrombosis in the leg (n = 15), and pulmonary thromboembo- with the development of arterial and venous thromboses. lism (n = 9). Patients younger than 3 y were 27% of all patients. Thromboembolism, formerly recognized as a rare event in The largest age group was <1 y of age n( = 50, 16.3%), and the children (1), has been increasingly diagnosed as a complica- number of other age groups were similar (median: 12, ranging: tion of sepsis, cancer, cardiovascular disease, and therapy- 9–17 (3.0–5.5%)). related events. Recent advances in the intensive cares, cardiac surgery, and transplantation medicine along with the imaging PC, PS, and AT Activities in Patients diagnosis may contribute to the increased number of pediatric A total of 122 patients had the reduced activity of either anti- patients with thrombosis (2,3). The established genetic risks coagulant; 95 with low PC (31%), 48 with low PS (16%), and 20 of venous thromboembolism (VTE) include protein C (PC), patients with low AT activity (7%; Figure 1). Low PC patients

1Comprehensive Maternity and Perinatal Care Center, Kyushu University Hospital, Fukuoka, Japan; 2Department of Pediatrics, Yamaguchi University Graduate School of Medicine, Ube, Japan; 3Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 4Department of Clinical Chemistry and Laboratory Medicine, Kyushu University Hospital, Fukuoka, Japan. Correspondence: Shouichi Ohga ([email protected]) Received 5 March 2015; accepted 29 June 2015; advance online publication 14 October 2015. doi:10.1038/pr.2015.180

Copyright © 2016 International Pediatric Research Foundation, Inc. Volume 79 | Number 1 | January 2016 Pediatric Research 81 Articles Ichiyama et al. were most frequently determined in the lowest age group (0–2 Mutations of PC, PS, and AT Genes in Patients y, 45%), while low PS or low AT patients were frequently found Of 62 children who showed the low level of at least one factor,­ in the highest age group (16–20 y; PS: 30% and AT: 20%). The mutation carriers were found in low PC (n = 8/39, 21%), PS number of patients with low activity in age groups are shown (n = 8/27, 30%), or AT (n = 2/5, 40%) patients, respectively. in Figure 2. The proportions of low PC patients were higher Forty-four patients carried no mutations. Age distribution than those of low PS or AT patients in age 0–2 (each <0.00001) of 62 patients differed among three deficiencies (0.0042; and 13–15 y (0.0001, 0.0155), respectively. The proportion of Figure 3). The proportion of age 0–2 y patients with PC defi- low PC or PS patients (19%) was each higher than that of low ciency (44%) was the highest among any age group patients AT patients in 7–12 y (0.0005). The number of patients show- with PS or AT deficiency. Six of eight patients with PC gene ing both low levels of PC and PS activities were 25 (20%), 5 of mutations (75%) were found in the age group 0–2 y, in which whom had the low levels of all three factors (Supplementary only two PS and no AT mutation carriers were determined. Figure S1 online). Four of eight patients with PS gene mutations (50%) were found in the age group 7–12 y. No ratio of mutated to analyzed patients differed in the age-groups.

Thromboembolism (June 1993–March 2012) Clinical Onset, Genotype, and Activity Levels of PC, PS, and AT in 1,867 patients 1,561 patients age ≥ 21 y, the Mutation Carriers Anomaly, inborn errors of metabolism Eighteen patients with a mutation are listed in Table 1. Seven 306 patients 16.4%, age 0–20 y out of eight PC-deficient patients suffered from intracranial Low plasma activity thromboembolism and five of them were younger than 2 y. On PC PS AT Normal activities of PC, PS, and AT: 184 the other hand, six of eight PS-deficient patients presented in 122 patients 95 48 20 the second decade of life, the onset of which were adult type Acquired thrombosis (including APS): 60 of pulmonary thromboembolism in five of them. Double allele (homozygous or compound het- 39 27 5 62 patients erozygous) and one allele (heterozygous) mutation(s) were Genetic study 31 19 3 found in half of eight PC-deficient patients, respectively. On 44 patients No mutation the other hand, double allele mutated patients were only two

882 Mutation of eight heritable PS-deficient patients, both of whom carried PS-Tokushima. Two other patients had PS-Tokushima. Both Figure 1. Flowchart of the genetic study of pediatric in patients with heritable AT deficiency carried heterozygote AT a Japanese institution. During the 20 y, 306 patients (≤20 y of age) were mutation. Four of eight patients with PC gene mutation and screened by the plasma activities of protein C (PC), protein S (PS), and anti- thrombin (AT) and underwent the genetic study if they have idiopathic or * unusual thrombophilic predispositions. ns ** ns Number of ‡ ns † ns patients ** ‡nsnsnsnsnsnsns 100 ** * PC deficiency

80 PS deficiency

60 1 6 1 AT deficiency Mutation 2 2 40 Mutation 4 Mutation 1 1 20

0 n = 39 n = 27 n = 5 PC PS AT PC PS AT PC PS AT PC PS AT PC PS AT 0–2 3–6 7–12 13–15 16–20 Figure 3. Age distribution of patients with thromboembolism carrying PC, PS, and AT gene mutation. The circle size represents the number of Figure 2. Proportion of patients who showed the low plasma activity of patients. (a) PC deficiency (n = 39): 0–2 y, 44%; 3–6 y, 13%; 7–12 y, 8%; PC, PS, and AT according to the age groups. 0–2 y: infants (n = 84; low PC, 13–15 y, 23%; and 16–20 y, 13%. Six of eight patients with PC gene muta- 45%; low PS, 8%; low AT, 5%), 3–6 y: preschoolers (n = 53; low PC, 14%; tions (75%) were found in the age group 0–2 y. (b) PS deficiency n( = 27): low PS, 8%; low AT, 4%), 7–12 y: elementary school children (n = 73; low 0–2 y, 15%; 3–6 y, 15%; 7–12 y, 33%; 13–15 y, 4%; and 16–20 y, 33%. Four of PC, 19%; low PS, 19%; low AT, 1%), 13–15 y: junior high school students eight (50%) patients were found in the age group 7–12 y. (c) AT deficiency (n = 41; low PC, 44%; low PS, 17%; low AT, 5%), 16–20 y: high school and (n = 5): 0–2 y, 20%; 3–6 y, 40%; 16–20 y, 40%. All AT gene mutation carriers college or university students (n = 55; low PC, 33%; low PS, 30%; low AT, were found in the age group more than 3 y. Closed areas indicate 0–2 y 20%). Closed bar represents the patients who showed each low plasma of age, open areas indicate 3–6 y of age, hatched areas indicate 7–12 y of activity. *P = 0.0155; **P = 0.0005,†P = 0.0001; ‡P < 0.00001. ns, no statisti- age, gray areas indicate 13–15 y of age, and dotted areas indicate 16–20 y cal significance. of age. *P = 0.0042, **P = 0.0019. ns, no statistical significance.

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Table 1. Plasma activity of protein C, protein S, and antithrombin and each gene mutation in pediatric patients Plasma Plasma activity (%) activity (%) mother/father Family Patient Sex Age Diagnosis PC PS AT Mutation history PC PS AT PC deficiency PROC

F 6 d Cerebral-VTE, <5 45 111 Ex8; c.887C>T, p.Leu265Phe Negative 63/63 69/99 125/110 twin-1 Ex9; c.1360G>C, p.Trp422Cys F 6 d Cerebral-VTE, <5 24 141 Ex8; c.887C>T, p.Leu265Phe Negative 63/63 69/99 125/110 twin-2 Ex9; c.1360G>C, p.Trp422Cys F 9 d ICH, PF <10 140 60 Ex9; c.1109G>A, p.Val339Met Negative 32/72 68/110 115/120 Ex9; c.1362delG, p.Gly423ValfxX82a F 13 d Cerebral VTE <5 29 110 Ex4; c.258delT, p.Leu55ArgfsX6 Negative 46/48 na/na na/na Ex9; c.905C>T, p.ArG271Trp M 4 mo Intrahepatic 25 91 104 Ex7; c.671_673delAAG, p.Lys193del Negative 64/93 67/95 97/101 F 1 y Meningitis, ICH/ 31 38 68 Ex4; c.356G>T, p.Asp88Tyr Positive 50/99 66/83 na/105 CI, PF F 14 y ICH, sinus 32 62 87 Ex6; c.603A>G, p.Asp170Gly na na na na thrombosis F 18 y CI lt. frontal 39 101 79 Ex3; c.293G>A, p.Glu67Lys na na na na PS deficiency PROS1 M 3.5 mo Thalamic 48 69 85 Ex6; c.927A>G, p.Lys196Gluc na na na na bleeding, lt. F 2 y Hypoplasia of 90 61 116 Ex6; c.927A>G, p.Lys196Gluc na na na na optic papilla lt. F 10 y DVT, epilepsy 60 10 na Ex2; c.418-1G>C, Int1 splice acceptor site na na/92 <10/84 na M 11 y DVT 5b 12 122 Ex15; c.2361-2delAA, p.Lys674GlufsX24 na na na na M 12 y DVT 10b 2 115 Ex1; c.391T>C , p.Leu17Pro Positive 102/na 54/na 114/na Ex6; c.927A>G, p.Lys196Gluc F 12 y IE, multiple CI 66 12 103 Ex13; c.1884C>T, p.Arg515Cys Positive na 69/low na F 16 y DVT 24 5 90 Ex13; c.1884C>T, p.Arg515Cys na 103/107 14/71 97/97 Ex6; c.927A>G, p.Lys196Gluc M 16 y , 21 11 105 Ex12; c.1692C>T, p.Arg451X na 119/101 29/129 109/116 PTE AT deficiency SERPINC1 F 4 y Vasculitis 121 74 14 Ex3; c.561T>C, p.Ser148Pro Positive 104/na 30d/na 58/na F 18 y Pulmonary 76 47 55 Ex7; c.1438T>C, p.Phe440Ser na 113/na 67/na 65/na infarction The italic formatting indicates low activity according to the conditional limits of age groups. CI, cerebral infarction; DVT, deep vein thrombosis; ICH, ; lt., left ; na, not assessed; PF, ; PTE, pulmonary thromboembolism; VTE, venous thromboembolism. aPC-Nagoya. bNo mutation of PROC. cPS-Tokushima. dAt 19 gestational weeks. seven of eight patients with PS gene mutation showed reduced pediatric thrombosis, and the age-dependent presentation of activities of PS and PC, respectively. each PC, PS, or AT deficiency before adult life. Congenital PC and PS deficiencies were the major thrombophilias in Japanese DISCUSSION children with different age distribution. Predominant cere- The first survey on PC, PS, and AT deficiency in Japanese bral thromboembolism in PC-deficiency contrasted sharply thromboembolic children revealed the contribution to 5.9% of with extracranial VTE in the other two deficiencies. The

Copyright © 2016 International Pediatric Research Foundation, Inc. Volume 79 | Number 1 | January 2016 Pediatric Research 83 Articles Ichiyama et al. genotype of PS-deficient children reflected the high prevalence (18,26), partly arising from the inherent variation of PC path- of PS-Tokushima in Japanese general population (1.1–1.8%; way (27,28). Clinical manifestation and thrombin generation Table 1) (12,13). The Israeli-German cohort studies (14–16) differ among the family members having the same PROC have recently reported the prevalence of congenital deficiencies mutation (29). Otherwise, absolute PC deficiency rather than PC (7.4%), PS (8.2%), and AT (6.6%) in pediatric patients with PS deficiency may contribute to the development of throm- thromboembolism aged 0.1–18 y. In the cohorts, the propor- boembolism in the newborn and young infants (30). Half of tion of neonates was also higher in PC deficiency (32%; 8/25 the PS gene–mutated patients carried PS-Tokushima, two of patients) than seen in PS deficiency (10%; 3/30 patients) or AT whom had compound heterozygous mutations with one allele deficiency (14%; 3/21 patients). Half of PC-deficient newborns PS-Tokushima (Table 1). Both patients lacked PS activity and presented cerebral sinovenous thrombosis and stroke. In this developed deep vein thrombosis at 12 and 16 y of age. These setting, PC-deficient infants characterized the phenotype of findings suggested that high allele frequency of PS-Tokushima pediatric thrombophilias (purpura fulminans and intracranial made an impact on the genotype of severe PS-deficient chil- lesions) beyond the ethnicity (17). The age-specific prevalence dren in Japan. By contrast, six of eight patients with PC defi- and phenotype of pediatric PC deficiency may be useful in the ciency presented in infancy, four of whom carried double allele genetic screening for pediatric thrombophilias. mutation (one PC-Nagoya) having undetectable plasma activ- The first concern is the applicability of the lower limits of PC, ity. The genotypes of pediatric PC and PS deficiency corrobo- PS, or AT activity in age groups for the genetic screening. The rated the previous studies on Japanese adult population. ranges of anticoagulant activities often vary until the attain- The other concern is the distinct affected site between ment of adult levels, reflecting the individual maturation and PC-deficient (intracranial lesion) and PS-deficient children vitamin K status (18,19). Our previous adult study assessed (extracranial VTE). Premature infants with perinatal compli- by the same measures reported that half of VTE patients with cations are liable to bleed in the brain. However, intracranial each low activity carried the heterozygous mutation, which lesions including sinovenous thrombosis were also observed resulted in 33% positive rate of all patients (20). Miyata et al. in adolescence (Table 1). The expression levels of endothelial (21) confirmed that 32% of adult VTE patients carried hetero- PC receptor and thrombomodulin are constitutively low in the zygous mutation of PC, PS, or AT, in the twice sample size, brain compared with other organs. In contrast to the basilar without the screening of plasma activity. In this setting, the arteries or choroid plexus, intracerebral vascular endothelium concordant mutation rate by the independent studies would does not express thrombomodulin (31). Free PS works as a warrant the utility of plasma activities for genetic screening cofactor of activated PC in the PC pathway. Circulating free in Japanese children. No mutation in >30% of patients raises PS molecules are relatively high in infancy because of physi- the possibility of large gene deletions, polymorphisms, and ologically low C4-binding protein levels (26). It may explain the other genetic variations or certain modifiers affecting the the preferential cerebral lesions in PC-deficient children. activity and antigen concentration (22). The present measures Activated PC and thrombomodulin have homeostatic signals studied polymorphisms including the promoter regions but critical in regulating , inflammation, endothelial not deletions. Large deletions are reported to be 3–6% in these barrier function, and neuroprotection (32). PC-deficient chil- deficiencies (5). Cooper et al. (23) revealed that large dele- dren might be vulnerable to the brain damage in association tions make up between 7 and 10% of PS and AT mutations with prothrombotic triggers. and only 1% of PC mutations. The rare occurrence of throm- One of the limitations in this report was that the results were bosis and the wide range of factor activities impeded to define obtained from a single center study. The number of muta- the standard values of “true healthy” children who develop no tion carriers was small even in the prolonged study. The other thromboembolism until the forties. In this study, there was no limitation was that the consumption effects and/or the plasma significant difference in the ratios of mutated patients to the replacement therapy might not be completely excluded in low PC patients who underwent the genetic analysis and in assessing the activity levels of the natural anticoagulants. The the ratios of mutated patients to the low PS patients analyzed shorter biological half-life of PC than that of PS or AT might in any age groups. Taking into account high safety margins, influence the dissociated low activity of PC in infants. However, the limits of factor activity might be practical for the genetic these findings do not explain the distinct presentation between screening of pediatric thrombophilias. PC deficiency in infancy and PS deficiency in adolescents or The proportion, genotype, and first presentation of PC, PS, young adults. Multiplex ligation-dependent probe amplifica- and AT deficiency during childhood were distinct from those tion and/or single-nucleotide polymorphism array are needed of adult patients. Patients with low PC activity were almost to search the deletion in the future study. Further compara- twice the number of those showing low PS activity (Figure 1). tive study on PC- and PS-deficient infants may shed some light On the other hand, the number of patients who carried either on the pathophysiology and optimal management of pediatric PROC or PROS1 mutation was the same eight. The first nation- thrombophilias. wide survey for pediatric thrombophilia in Japan suggested a In conclusion, PC gene–mutated patients mostly presented higher prevalence of PC deficiency than expected (24,25). with intracranial thromboembolism less than 2 y of age. On The discrepancy may be explained by the wider range of PC the other hand, PS/AT gene–mutated patients were prefer- activity than that of PS or AT activity in infant and children entially found in school age children with extracranial VTE.

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The genetic screening of pediatric thrombophilias could be STATEMENT OF FINANCIAL SUPPORT This study was supported by a grant from the Kyushu University Clinical effectively targeted to stroke in low PC infants and deep vein Research Network Project and by a grant from the Ministry of Health, Labour thrombosis in low PS /AT school age children. and Welfare of Japan. Disclosure: The authors have no conflict of interests to disclose. METHODS Patients and Screening Protocol References From 1993 to 2012, 1,867 patients with thrombosis in Kyushu 1. Spentzouris G, Scriven RJ, Lee TK, Labropoulos N. Pediatric venous University and affiliated institutions were consecutively assessed thromboembolism in relation to adults. J Vasc Surg 2012;55:1785–93. for thrombophilic predispositions (Figure 1). After the exclusion of 2. Nowak-Göttl U, Janssen V, Manner D, Kenet G. Venous thromboembo- patients with anomaly and inborn errors of metabolism, 306 patients lism in neonates and children–update 2013. Thromb Res 2013;131:Suppl aged less than 21 y of age (16.4%) were enrolled for the study. Clinical 1:S39–41. information of thromboembolic events and/or was 3. Tolbert J, Carpenter SL. Common acquired causes of thrombosis in chil- collected from the medical records and the interviews of attending doctors. Of 122 patients showing the low plasma activity, 60 were dren. Curr Probl Pediatr Adolesc Health Care 2013;43:169–77. excluded because of acquired thrombotic disorders including vas- 4. Caspers M, Pavlova A, Driesen J, et al. Deficiencies of antithrombin, pro- culopathy, antiphospholipid syndrome, and autoimmune diseases. tein C and protein S - practical experience in genetic analysis of a large Finally, 62 patients underwent the genetic analysis because they had patient cohort. Thromb Haemost 2012;108:247–57. no explainable causes of thromboembolism other than PC, PS, or AT 5. Roberts LN, Patel RK, Arya R. Venous thromboembolism and ethnicity. Br deficiency. Informed consent was obtained from parents of children J Haematol 2009;146:369–83. aged <21 y of age. This study was certified by the Institutional Review 6. Shatla HM, Tomoum HY, Elsayed SM, et al. Inherited thrombophilia in Board of Kyushu University (Fukuoka, Japan; #232-02, #448-00). pediatric ischemic stroke: an Egyptian study. Pediatr Neurol 2012;47: 114–8. Coagulation Study 7. Sirachainan N, Sasanakul W, Visudibhan A, Chuansumrit A, Wongwer- To exclude the consumption effects at the diagnosis of thromboem- awattanakoon P, Parapakpenjune S. Protein C deficiency in Thai children bolism, the antigen/activity levels of PC, PS, and AT were repeat- edly assessed when the clinical conditions were stabilized a couple of with thromboembolism: a report of clinical presentations and mutation weeks to months after the onset of thromboembolism. The measure- analysis. Thromb Res 2010;125:200–2. ment of anticoagulation factors were performed as described previ- 8. Shen MC, Lin JS, Tsay W. Protein C and protein S deficiencies are the ously (20,33). Anticoagulant activities of PC and PS were determined most important risk factors associated with thrombosis in Chinese venous using the Staclot PC kit and the Staclot PS kit (Diagnostica Stago, thrombophilic patients in Taiwan. Thromb Res 2000;99:447–52. Asnieres, France), respectively. A chromogenic substrate was used 9. Tang L, Jian XR, Hamasaki N, et al. Molecular basis of to assay for AT activity as heparin-dependent inhibition of bovine in China. Am J Hematol 2013;88:899–905. thrombin (Chromostrate ATIII kit; Hitachi, Tokyo, Japan). The adult 10. Holzhauer S, Goldenberg NA, Junker R, et al. Inherited thrombophilia in references were first standardized by using pooled normal plasma, in children with venous thromboembolism and the familial risk of thrombo- which a level below 3 SD was defined as reduced activity. According embolism: an observational study. Blood 2012;120:1510–5. to the data in Japanese children (18,26), the lower limits of each activ- 11. Sosothikul D, Kittikalayawong Y, Aungbamnet P, Buphachat C, Seksarn P. ity were defined as follows: in age <90 d: PC 60%, PS 60%, and AT Reference values for thrombotic markers in children. Blood Coagul Fibri- 65% of the adult reference levels (PC 75%, PS 60%, and AT 80%); 90 nolysis 2012;23:208–11. d–2 y: PC 85%, PS 85%, AT 65% of the adult levels; 3–6 y: each 85% 12. Sakata T, Okamoto A, Mannami T, Tomoike H, Miyata T. Prevalence of of adults; and 7–20 y: same as the adults (Supplementary Table S1 protein S deficiency in the Japanese general population: the Suita Study. online). J Thromb Haemost 2004;2:1012–3. Gene Analysis of PC, PS, and AT 13. Ikejiri M, Wada H, Sakamoto Y, et al. The association of protein S Genomic DNA was extracted from peripheral blood leukocytes. Tokushima-K196E with a risk of deep vein thrombosis. Int J Hematol Direct sequencing of PC (PROC exon 1–9), PS (PROS1 exon 1–15), 2010;92:302–5. and AT (SERPINC1 exon 1–6) genes was performed as previously 14. Limperger V, Klostermeier UC, Kenet G, et al. Clinical and laboratory described (20). The exon and exon–intron boundary regions of each characteristics of children with venous thromboembolism and protein gene including promoter region were amplified by PCR, and the prod- C-deficiency: an observational Israeli-German cohort study. Br J Haematol ucts were then subjected to direct sequencing using ABI 377 (Perkin 2014;167:385–93. Elmer Applied Biosystems, Foster City, CA). 15. Klostermeier UC, Limperger V, Kenet G, et al. Role of protein S deficiency Statistical Analysis in children with venous thromboembolism. An observational interna- The median of continuous variables was assessed by the Mann– tional cohort study. Thromb Haemost 2015;113:426–33. Whitney U-test. The distribution difference of countable variables 16. Limperger V, Franke A, Kenet G, et al. Clinical and laboratory character- was analyzed by chi-square test or Fisher’s exact test. P values less than istics of paediatric and adolescent index cases with venous thromboem- 0.05 were considered significant. bolism and antithrombin deficiency. An observational multicentre cohort study. Thromb Haemost 2014;112:478–85. SUPPLEMENTARY MATERIAL 17. Price VE, Ledingham DL, Krümpel A, Chan AK. Diagnosis and man- Supplementary material is linked to the online version of the paper at http:// agement of neonatal purpura fulminans. Semin Fetal Neonatal Med www.nature.com/pr 2011;16:318–22. 18. Ignjatovic V, Mertyn E, Monagle P. The coagulation system in children: ACKNOWLEDGMENTS developmental and pathophysiological considerations. Semin Thromb We thank Michiyo Urata, Takeshi Uchiumi (Department of Clinical Chemistry Hemost 2011;37:723–9. and Laboratory Medicine, Kyushu University Hospital, Fukuoka, Japan), and 19. Bereczky Z, Kovács KB, Muszbek L. Protein C and protein S deficiencies: Naotaka Hamasaki (Department of Clinical Chemistry, Faculty of Pharma- similarities and differences between two brothers playing in the same ceutical Sciences, Nagasaki International University, Nagasaki, Japan) for the game. Clin Chem Lab Med 2010;48:Suppl 1:S53–66. establishment of system, and all the staffs of the Department of Pediatrics, 20. Kinoshita S, Iida H, Inoue S, et al. Protein S and protein C gene mutations Kyushu University Hospital, Fukuoka, Japan for the clinical managements. in Japanese deep vein thrombosis patients. Clin Biochem 2005;38:908–15.

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